Since the advent of biotechnology many protein therapeutics have been identified as having significant therapeutic benefits for many potentially serious diseases. Such diseases include cancer, heart disease, diseases of the central nervous system, diseases of the immune system, diseases of the blood and circulatory system, and many hormonally based diseases. Unfortunately, many of these medical disorders are chronic and require continuous administration of the required therapeutic.
However, the broad utilization of protein therapeutics, and many other hard to deliver therapeutics depends on the ability to easily administer them to patients in a controllable and acceptable manner. Clearly oral administration would be the most desirable method for administering such materials to patients who must take them for extended periods of time. Unfortunately, the ability to administer proteins in this fashion has never been achieved because typically proteins and peptides are easily metabolized in the stomach and intestine when introduced orally to patients. In addition to problems related to degradation, their molecular weights are typically too high to allow for significant transport across the gut wall into the circulatory system. This is true even if they can be protected from extensive degradation prior to getting to the gut wall barrier. As a result protein therapeutics can only be given by injection (typically intravenous (IV) or subcutaneous), and this has severely restricted their broad utilization for many diseases.
A typical and very important example of a potential application for an oral protein delivery technology is the treatment of diabetes. There are currently more than 50 million people around the world who suffer from one of several well known forms of diabetes. In has been known that most forms of diabetes can be treated with regular doses of insulin administered to counter the buildup of blood glucose which takes place when diabetics ingest foods. In addition to the patient acceptability problem associated the delivery of insulin by injection, there are other serious difficulties associated with providing insulin by injection. These include supplying and using the necessary syringes and other medical devices to patients who suffer from one form or another of diabetes in many parts of the world, the inappropriate time profile associated with delivery by injection, and the excessive amounts of insulin to which various organs of the body are subjected when insulin is administered in this fashion. All of these problems would be ameliorated by the development of an oral form of insulin which could be administered, and made available in the circulatory system along with food.
Because diabetes is a common disease, the properties and effects of Insulin are well known. It is a polypeptide hormone having a molecular weight of about 6000. It influences many metabolic processes in humans and other animals. For example, it increases the diffusion of glucose into living tissues as well as its use by cells in metabolic processes. It also reduces the glycogen content in the liver and increases its concentration in muscles. Insulin also increases protein synthesis rates, and affects other metabolic processes.
Indeed, an approach which would permit the direct oral delivery of proteins and peptides has been sought for many years, as it has been generally recognized that such an approach, if developed, would have important advantages and thus greatly expand the potential uses for protein and peptide therapeutics. Nevertheless, and in spite of extensive efforts to develop insulin preparations resistant to the action of digestive proteinase and capable of delivery into the blood stream through the intestinal mucosa. No such approach has been found and used for the general administration of insulin or other protein or peptide therapeutics up to now. Extensive work continues today throughout the world in spite of repeated failures. With respect to protein and peptide therapeutics produced by biotechnology, oral delivery has remained one of the major unfulfilled goals for at least the last 15 years. Unfortunately no effective method to accomplish this has yet been found, for insulin or any other protein or peptide. The invention and approaches described herein address this critical, and as yet unresolved issue, and provide a direct pathway to the potential practical development of an oral delivery methodology for proteins and peptides, and other hard to deliver therapeutics.
Unfortunately, the principle effective method accepted for administering insulin to diabetic patients is still via subcutaneous (SQ) and intramuscular (IM injection of a preparation containing insulin. Dosage amounts, and administration profiles vary widely from patient to patient, depending on many factors such as body weight and size, the nature of the specific form and severity of diabetes in each patient, and the degree of diet control which can be exercised by each patient. It is not possible to identify a single dosage composition or amount which can be considered a standard human dosage form. Typically such treatments must be tailored for each patient to produce a given biologic and therapeutic profile and injections must be repeated several times daily for most patients. The need to inject insulin repeatedly (500 to more than 1000 times per year for many patients) results in physical pain, emotional distress, and many related problems for patients who must undergo this treatment on a regular basis. For this reason many marginal patients, or patients who do not live in areas of the world where syringes and needles are not easily accessible do not receive therapy.
Indeed, the disadvantages of administration by injection of most protein therapeutics, has been one of the principle elements limiting the use of proteins, as therapeutics for chronic diseases. The same problems and limitations apply to the use of proteins in animals where multiple doses would be required to achieve the necessary biological effect, and to non-protein therapeutics which are not orally bioavailable.
The compositions and method of this invention relates to any number of potentially efficacious proteins and peptides as well as other therapeutics. The oral delivery of insulin, and growth hormones are used to illustrate the general applicability of the methodology and the formulations described. We show animal studies, formulations, and a methodology for making the formulations which permits the creation of insulin preparations resistant to the action of digestive proteinase. The use of these formulations permits the contained therapeutic protein to penetrate the intestinal mucosa and enter the circulatory system where it can manifest its therapeutic effect. We show further that therapeutic efficacy can be achieved with dosage levels of insulin which are comparable to the dosage levels commonly used in conventional therapeutic injections.
Several approaches have been proposed by others in the past to achieving the oral delivery of insulin. One such approach involves the modification of the insulin molecule itself. Another such approach involves the modification of insulin by replacing for example, the C-end residue of threonine with a more stable glycine residue. Yet another approach involves the hydrophobization of the insulin molecule and yet other such approaches involves the preparation of monosaccharide derivatives of insulin and the binding of insulin to other proteins.
However, despite the fact that in many of these attempts the modification of the insulin molecule does result in an increase in insulin resistance to the action of proteinase in vitro, or has been shown in some cases to result in prolonging its effect in the body when injected IV or IM, effective oral formulations have never been demonstrated using these methods. The goal of general oral administration of insulin (and most other peptide therapeutics and generally non-orally available biologically active agents) remains unfulfilled.
Another approach to solving this problem which recently has been reported in the literature involves administering insulin in combination with compounds increasing the penetrability of insulin through the intestinal wall. This approach is intended to facilitate the penetration of the active agent through the intestinal barrier, and into the blood stream. This approach is also potentially applicable for other proteins as well as to insulin since the primary action of the added agent is on the physiology, permeability and penetrability of the intestinal barrier. Fatty acid salts, surfactants, bile salts (cholate), chelate-forming compounds are all examples of materials which have been proposed and for which experiments have been attempted in this regard.
In principle the increased permeability of the intestinal wall induced by such added materials should contribute to an increased amount of protein reaching the blood stream in an active state. However, up to the present time, effects have been observed only with mixtures of such materials administered directly into the intestines, by-passing the esophagus and stomach. This approach (often referred to as peroral), has not shown a therapeutic effect even when high doses on insulin or other proteins and peptides are used. Even had positive therapeutic effects been observed, this form of administration is extremely difficult, and potentially uncomfortable for a human patient. It is not an acceptable answer to the generic problem of protein delivery or even the treatment of very severe diabetes. Yet another disadvantage of such formulations as have been studied in this manner are the potential long term effects of permeation enhancers on the intestines. Still another disadvantage is that such approaches are non-specific and can result in many undesirable materials and antigenic compounds normally excluded from transport (including viruses, degrading enzymes, and other toxic materials normally found in the intestine) transporting into the blood stream. Finally, it is clear that such approaches do not protect the formulation, including the therapeutic effect, from the degrading action of proteinase and/or other chemically active agents and enzymes present in the stomach and the gullet.
While one could modify such approaches to include in the formulations protective protease inhibitors, which could theoretically protect insulin to some degree, such work has not been carried out and shown to be effective in a formulation intended for conventional oral delivery. A similar (not very useful) result has been obtained when compositions containing protease inhibitors, insulin and compounds increasing the penetrability of intestinal walls were combined and administered directly to the bowels. In fact such an approach, with the long term effect of permeation enhancers on the intestinal and bowel wall functionality, could be very damaging.
One of the more common experimental approaches to creating oral forms for insulin delivery has been to place the insulin inside a protective shell which protects the active agent as it is passing through the digestive track up to its entry into the small intestine. The protective shell is designed to disintegrate in the small intestine releasing active insulin (or other agent if the approach is used with other peptides or proteins). As such, various coating polymers have been used with solubility properties tailored to permit the coating shell to dissolve within the small intestines. This can be accomplished by selecting coating formulations with enhanced solubility at pH's appropriate to the small intestine. In addition, microparticulates such as liposomes, hydrogels, nanocapsules and specifically biodegradable polymers (nanoparticles) have been investigated. However for all the work which has been addressed to this issue, there are no effective delivery systems or formulations for the oral administration of any protein, much less insulin or growth hormone.
Injectable insulin-containing compositions have also been reported (U.S. Pat. No. 5,049,545) which are comprised of insulin immobilized on a polymer (including a polymer hydrogel). The polymers used in such compositions are represented by materials such as starch, dextran, polyoxyethylene, polyvinylpyrrolidone, cross-linked collagen, non-therapeutically active proteins and derivatives thereof, and these formulations have included inhibitors of proteolytic enzymes. This approach has resulted in the insulin-containing polymers displaying an increased resistance to the effect of blood proteolytic enzymes, and has been shown to yield formulations which show an increased duration of insulin activity when administer directly into the bloodstream. However, the insulin-containing polymer compositions synthesized in this work do not show significant stability to the attack of the digestive enzymes and hence are not useful for oral delivery.
Saffran M., Kumar G. S., et al. Biochem. Soc. Trans. 1990, v.18, N. 5,P.752 have shown and reported on insulin-containing polymer compositions comprising an insulin-containing gelatin capsule coated by a copolymer of styrene with hydroxyethylmethacrylate, said polymer then being crosslinked with a divinylbenzene azo-containing derivative. On oral administration, the crosslinked copolymer is degraded by the action of microorganisms within the intestines with the release of insulin, a small amount of which is shown to penetrate the intestinal wall. However, a disadvantage of these compositions is that they show a low resistance to the action of digestive enzymes and hence only a very small amount of insulin is demonstrated to actually pass through the intestinal wall and appear as active insulin in the circulatory system. With the oral administration of the above-mentioned crosslinked polymer into rats in an amount of from 1 to 40 mg per rat (as calculated for insulin), the maximum reduction of glucose concentration in animal blood was 25% on average (from 384 mg/100 ml to 287 mg/100 ml) and was observed 3-4 hours after administration of the preparation. This is far below the desirable reduction of blood glucose due to insulin, and demonstrates the general lack of effectiveness of this approach. Also this approach has not been shown to be useful for the administration of any other protein or peptide, nor for use in treating humans.
Damge, C., J Controlled Release, 1990, V. 13. P. 233, showed that spherical nanocapsules may be produced from biodegradable polyisobutyl-2--cyanoacrylate, said nanocapsules having a diameter of from 250 to 350 nm, said nanocapsules containing insulin dispersed in a lipid phase. However, upon oral administration of these nanocapsules, very little effect on blood glucose was observed when normal therapeutic levels of insulin were contained within them. A measurable (though not very great) reduction in glucose concentration of only 25% was observed even when very high insulin doses were used compared to generally accepted therapeutic doses. Indeed the doses used in this study were in the range of 100 U/kg (almost 20 times higher than doses typically injected into humans to achieve therapeutic efficacy). Even more significant from a therapeutic standpoint, the insulin in this demonstration was shown to be measurably active 6 days after the capsules were administered to the host animal. Such results throw into serious question the significance of the experiments since there is no therapeutically acceptable mechanism by which the delivered protein could remain the intestine of the test animals for such a period of time undigested and unmodified (complete elimination being typically effected in 12-24 hours). Even if there were an adequate explanation for these results, such a dosage level, as well as the delivery and therapeutic profile associated with this approach, is considered to be unacceptable for insulin, and indeed for most protein therapeutics whose administration and therapeutic effect must follow closely one upon the other. In particular for insulin it is desirable for this hormone to move across the intestinal wall and enter the circulatory system along with the absorbed nutrients from food which may be ingested by the host. Such a profile is clearly not associated with the work reported by Damge.
Greenley R. Z., Broun T. M. et al. Polymer Prepr., 1990, v. 31, N 2. P.182. have reported a composition which is similar in some aspects to the compositions claimed herein. In their composition insulin is immobilized within the volume of a crosslinked polymer modified with an inhibitor of proteolytic enzymes. The crosslinked polymer is substantially a polyacrylic or polymethacrylic acid polymer crosslinked with triethyleneglycoldi(meth)acrylate. The inhibitor is an aprotinin-protease inhibitor. A disadvantage of this composition is that the synthesized polymer hydrogels have little resistance to the action of digestive enzymes. As in the other studies reported above, this limitation results in a low measured activity of insulin passing through the intestinal wall and entering the circulatory system. It would certainly result in a similar limitation with respect to other proteins and peptides contained therein. Thus, when such a modified insulin formulation of the type described by Greenly et.al. was administered to rabbits in amounts corresponding to 50 IU/Kg, the concentration of glucose in the blood is reduced after 30 min from 380 to 360 mg/100 ml. After 300 min blood glucose is shown to reach 460 mg/100 ml (no therapeutic effect). In the same study the oral administration of an unmodified hydrogel with no inhibitor (containing 50 units of insulin) was accompanied by approximately a 23% reduction in blood glucose (from 310 to 240 mg/100 ml after 300 min). At the same time the subcutaneous injection of the rabbits with only 0.23 units of insulin leads to a reduction of blood glucose concentration from 330 to 120 mg/100 ml (insulin is reduced by 74%) 150 min after the injection. Again this experiment demonstrates the limited effectiveness in animal tests of formulations intended for oral administration of insulin and other similar proteins and peptides. It should be emphasized that even were such approaches successful. Indeed, the results reported by Greenley et. al. indicate that the oral formulation described in this study is somewhere between 200 to 600 times less effective on a dosage basis than normally injected insulin, and the therapeutic usage of such a dosage form would require that massive doses of insulin be fed to patients to achieve any therapeutic effect. This is not feasible from either a practical or an economic standpoint, even if the effects were in fact shown to extend to human patients, which is not the case in this study. The study further shows that simply administering a hydrogel-like material containing an enzyme inhibitor is not sufficient to cause therapeutic amounts of insulin or proteins to pass the intestinal barrier, and that the formulations of the invention described below are distinct in their nature and effect from those tested in the past.